Application of protein OsAA2 in regulation and control of heat resistance of rice

Abstract

The invention discloses application of protein OsAA2 in regulation and control of rice heat resistance. The amino acid sequence of the protein OsAA2 is as shown in SEQ ID NO: 2. Experiments prove that the heat resistance of rice can be improved by improving the expression quantity of the protein OsAA2 in Nipponbare rice; the improvement of the heat resistance shows that after high-temperature treatment, the length of the overground part and the root length are remarkably increased, and the content of malondialdehyde and the relative conductivity are remarkably reduced. The protein OsAA2 can regulate and control the heat resistance of rice. The method has an important application value.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of protein OsAA2 in regulating the heat resistance of rice. Background Art

[0002] Climate change is one of the important factors affecting the geographical distribution and growth and development of plants. The increasing severity of high temperature stress seriously threatens agricultural production and causes a reduction in grain production. High temperature affects the physiological and biochemical reactions of plants, causing changes in the internal structure of chloroplasts, reducing the content of photosynthetic pigments, and seriously inhibiting plant photosynthesis. People have conducted a lot of research on the mechanism of plant response to high temperature stress from the perspectives of physiology, biochemistry, metabolism, ecology, genetics, and evolution, and have accumulated rich data. Especially with the development of molecular biology, people can understand the mechanism of plant resistance to high temperature stress at the molecular level such as gene composition, expression regulation and signal transduction, and open up new ways to improve the stress resistance of plants by genetic engineering. Due to the complexity of plant stress resistance traits, it is very difficult to improve plant stress resistance by traditional breeding methods. With the development of molecular biology, genetic engineering has opened up new ways for plant stress resistance breeding, but the isolation of efficient stress resistance genes has become the main factor limiting plant stress resistance genetic engineering.

[0003] Rice ( Oryza sativa L.) is a major food crop that is sensitive to heat stress and is eaten by more than half of the country's population. Under heat stress, rice seedlings grow slowly, are stunted, wilt, and eventually die. Plants have evolved complex molecular networks that allow them to accurately sense changes in their surroundings and alter their cellular metabolism to better adapt to the environment. The discovery of heat-resistant genes is the cornerstone of rice molecular breeding, and the cultivation of heat-resistant rice varieties is an important measure to ensure national food security. Therefore, there is an urgent need to cultivate heat-resistant rice varieties to reduce food losses caused by heat stress. Summary of the invention

[0004] The purpose of the present invention is to improve the heat resistance of rice.

[0005] The present invention firstly protects the application of protein OsAA2, which can be S1) or S2): S1) Regulating heat tolerance in rice; S2) Breeding transgenic rice with altered heat tolerance.

[0006] The present invention also protects the use of nucleic acid molecules encoding protein OsAA2, which can be S1) or S2): S1) Regulating heat tolerance in rice; S2) Breeding transgenic rice with altered heat tolerance.

[0007] In any of the above applications, the regulation of heat tolerance of rice may be to increase the heat tolerance of rice or to decrease the heat tolerance of rice.

[0008] In any of the above applications, the cultivation of transgenic rice with altered heat resistance may be cultivation of transgenic rice with improved heat resistance or cultivation of transgenic rice with reduced heat resistance.

[0009] The present invention also protects a method for cultivating transgenic rice beta, which may include the following steps: increasing the expression level and / or activity of protein OsAA2 in the starting rice to obtain transgenic rice beta; compared with the starting rice, the heat resistance of the transgenic rice beta is improved.

[0010] In the above method, the expression level and / or activity of the protein OsAA2 in the starting rice can be increased by methods well known in the art such as transgenic, multiple copies, changing promoters, regulatory factors, etc., to achieve the effect of increasing the expression level and / or activity of the protein OsAA2 in the starting rice.

[0011] In the above method, increasing the expression level and / or activity of the protein OsAA2 in the starting rice can be achieved by introducing a nucleic acid molecule encoding the protein OsAA2 into the starting rice.

[0012] In the above method, the introduction of the nucleic acid molecule encoding the protein OsAA2 into the starting rice can be achieved by introducing a recombinant vector into the starting plant; the recombinant vector can be a recombinant plasmid obtained by inserting the nucleic acid molecule encoding the protein OsAA2 into an expression vector. The expression vector can specifically be a pCAMBIA1300 vector.

[0013] In the above method, the recombinant vector can be specifically a recombinant plasmid pCAMBIA1300- OsAA2 The recombinant plasmid pCAMBIA1300- OsAA2 Specifically, the restriction endonuclease of pCAMBIA1300 vector Bam The small DNA fragment between HI and XbaI was replaced with the DNA molecule shown in SEQ ID NO: 1 to obtain a recombinant plasmid.

[0014] The transgenic rice A can specifically be OE7, OE8 or OE11 mentioned in the examples; in this case, the starting rice is the rice variety Nipponbare.

[0015] The present invention also protects a method for cultivating transgenic rice B, which may include the following steps: inhibiting the expression level and / or activity of protein OsAA2 in the starting rice to obtain transgenic rice B; compared with the starting rice, the heat resistance of transgenic rice B is reduced.

[0016] In the above method, the expression level and / or activity of the protein OsAA2 in the starting rice can be inhibited by methods well known in the art such as DNA insertion, RNA interference, homologous recombination, and gene site editing to achieve the purpose of inhibiting the expression level and / or activity of the protein OsAA2.

[0017] In the above method, the inhibition of the expression level and / or activity of the protein OsAA2 in the starting rice can be achieved by introducing a substance that inhibits the expression of a nucleic acid molecule encoding the protein OsAA2 into the starting rice.

[0018] In the above method, the substance that inhibits the expression of the nucleic acid molecule encoding the protein OsAA2 can be a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule; The specific DNA molecule may include a sense segment, an antisense segment, and a spacer segment between them; The sense fragment may be a DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end; The antisense fragment may be the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 at positions 497 to 926 from the 5' end.

[0019] The specific DNA molecule may specifically consist of the sense segment, the antisense segment and a spacer segment located between them.

[0020] The recombinant plasmid containing the specific DNA molecule can be specifically a recombinant plasmid pTCK303- OsAA2 The recombinant plasmid pTCK303- OsAA2 Specifically, the restriction endonuclease of pTCK303 vector Spe I and Sac I recognition sequence between the small DNA fragments replaced by nucleotide sequence SEQ ID NO: 1 from 497 to 926 from the 5' end of the DNA molecule, restriction endonuclease Kpn I and Bam The small DNA fragment between the HI recognition sequences is replaced with a DNA molecule having a nucleotide sequence of SEQ ID NO: 3 (the DNA molecule shown in SEQ ID NO: 3 is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 from positions 497 to 926 from the 5' end), to obtain a recombinant plasmid.

[0021] The transgenic rice B can specifically be RNAi7, RNAi8 or RNAi21 mentioned in the examples; in this case, the starting rice is the rice variety Nipponbare.

[0022] The present invention also protects the rice breeding method A or the rice breeding method B.

[0023] The rice breeding method A protected by the present invention may include the following steps: increasing the content and / or activity of the protein OsAA2 in rice, thereby improving heat resistance.

[0024] The rice breeding method B protected by the present invention may include the following steps: reducing the content and / or activity of the protein OsAA2 in rice, thereby reducing heat resistance.

[0025] The present invention also protects substances that inhibit the expression of nucleic acid molecules encoding protein OsAA2.

[0026] Any of the above substances that inhibit the expression of the nucleic acid molecule encoding the protein OsAA2 may be a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule; The specific DNA molecule may include a sense segment, an antisense segment, and a spacer segment between them; The sense fragment may be a DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end; The antisense fragment may be the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 at positions 497 to 926 from the 5' end.

[0027] The specific DNA molecule may specifically consist of the sense segment, the antisense segment and a spacer segment located between them.

[0028] Any of the above-mentioned recombinant plasmids containing the specific DNA molecule can be specifically a recombinant plasmid pTCK303- OsAA2 The recombinant plasmid pTCK303- OsAA2 Specifically, the restriction endonuclease of pTCK303 vector Spe I and Sac I recognition sequence between the small DNA fragments replaced by nucleotide sequence SEQ ID NO: 1 from 497 to 926 from the 5' end of the DNA molecule, restriction endonuclease Kpn I and Bam The small DNA fragment between the HI recognition sequences is replaced with a DNA molecule having a nucleotide sequence of SEQ ID NO: 3 (the DNA molecule shown in SEQ ID NO: 3 is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 from positions 497 to 926 from the 5' end), to obtain a recombinant plasmid.

[0029] The present invention also protects the use of any of the above-mentioned specific DNA molecules, an expression box containing any of the above-mentioned specific DNA molecules, or a recombinant plasmid containing any of the above-mentioned specific DNA molecules in reducing the heat resistance of rice or cultivating transgenic rice with reduced heat resistance.

[0030] Any of the above-mentioned rice can be the rice variety Nipponbare.

[0031] The amino acid sequence of any of the above-mentioned proteins OsAA2 may be shown as SEQ ID NO: 2. SEQ ID NO: 2 consists of 317 amino acid residues.

[0032] Any of the above-mentioned proteins OsAA2 can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

[0033] Any of the above-mentioned nucleic acid molecules encoding protein OsAA2 may be a DNA molecule as shown in b1) or b2): b1) the coding region is the DNA molecule shown in SEQ ID NO: 1; b2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO: 1.

[0034] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0035] Wherein, SEQ ID NO: 1 consists of 954 nucleotides, and the nucleotides of SEQ ID NO: 1 encode the amino acid sequence shown in SEQ ID NO: 2.

[0036] Any of the above-mentioned improvements in heat resistance can be manifested as a significant increase in the length of the aboveground part, a significant increase in the root length, a significant decrease in the malondialdehyde content and / or a significant decrease in the relative conductivity of the rice plants after treatment with high temperature (above 42°C, such as 50°C, 51°C) compared to the starting rice plants.

[0037] Any of the above-mentioned reductions in heat resistance may be manifested as a significant reduction in the length of the aboveground part, a significant reduction in the root length, a significant increase in the malondialdehyde content and / or a significant increase in the relative conductivity of the rice plants after treatment with high temperature (above 42°C, such as 50°C, 51°C) compared to the starting rice plants.

[0038] Experiments have shown that increasing the expression of protein OsAA2 in Nipponbare rice can improve the heat resistance of rice; the improvement of heat resistance is manifested in that after high temperature treatment, the length of the aboveground part and the root length increase significantly, and the malondialdehyde content and relative conductivity decrease significantly. Protein OsAA2 can regulate the heat resistance of rice. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Picture 1 is the PCR amplification product in Example 1 OsAA2 Agarose gel electrophoresis results of full-length gene cDNA.

[0040] Picture 2 The real-time fluorescence quantitative PCR method for detecting transgenic rice in step 4 of Example 2 is as follows: OsAA2 Relative expression levels of genes.

[0041] Picture 3 It is the growth status of the rice seedlings to be tested in step 5 of Example 2.

[0042] Picture 4 It is the statistical result of the above-ground length and root length of the rice seedlings to be tested in step 5 of Example 2.

[0043] Picture 5 These are the measurement results of the malondialdehyde content and relative conductivity of the rice seedlings to be tested before and after the heat treatment in step 5 of Example 2. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0046] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0047] The pTCK303 vector is described in the following literature: Jiang J, Li J, Xu Y, Han Y, Bai Y, Zhou G, Lou Y, Xu Z, Chong K. RNAi knockdown of Oryza sativa root meander curling gene led toaltered root development and coiling which were mediated by jasmonic acidsignalling in rice. Plant Cell Environ.2007, 30(6):690-9. The pCAMBIA1300 vector is described in the following literature: Gou M, Su N, Zheng J, Huai J, Wu G, Zhao J, He J, Tang D, Yang S, Wang G. An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis . Plant J.2009 Dec;60(5):757-70. The specific method of transforming recombinant Agrobacterium into Nipponbare is referred to the following literature: Hiei Y, Ohta S, KomariT, KumashiroT. Efficient transformation of rice ( Oryza sativa L. mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. PlantJ.1994;6(2):271-82.Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H.Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J.2006;47(6):969-76. The method for determining the content of malondialdehyde is described in the following literature: Zhao Q, Zhou L, Liu J, Cao Z, Du X, Huang F, Pan G, Cheng F. Involvement of CAT in the detoxification of HT-induced ROS burst in rice anther and its relation to pollen fertility. Plant Cell Rep. 2018; 37(5): 741-757. The method for determining relative conductivity is described in the following literature: F. He, C. Gao, G. Guo, J. Liu, Y. Gao, R. Pan, Y. Guan, J. Hu, Maize annexin genes ZmANN33 and ZmANN35 encodeproteins that function in cell membrane recovery during seed germination, J. Exp. Bot. 70 (2019) 1183–1195. Gao, SX; Song, T.; Han, JB; He, ML; Zhang, Q.; Zhu, Y.; Zhu, ZGA calcium-dependent lipid binding protein, OsANN10, is anegative regulator of osmotic stress tolerance in rice. Plant Sci. 2020, 293,110420. Example 1. The coding gene of protein OsAA2 (i.e. OsAA2 Cloning of genes 1. The total RNA from the leaves of the rice variety Nipponbare (hereinafter referred to as Nipponbare) grown for 7 days was extracted by the Trizol method, and then the first-strand cDNA was reverse transcribed using reverse transcriptase, and then the ds-cDNA was synthesized using the SMART method to obtain the ds-cDNA of Nipponbare.

[0048] 2. Using the ds-cDNA of Nipponbare obtained in step 1 as a template, use primers OsAA2 -F: 5'-ATGGCGACGCTCACCGTCCC-3' and primer OsAA2 PCR amplification was performed using a primer pair consisting of -R: 5'-TCACTGCTCCTGACCCAGAAGA-3' to obtain a PCR amplification product.

[0049] The PCR amplification products were subjected to 1% agarose gel electrophoresis.

[0050] The results of 1% agarose gel electrophoresis are shown in Picture 1 (1 is the PCR amplification product).

[0051] 3. Recover a 954 bp DNA fragment from the PCR amplification product obtained in step 2.

[0052] The DNA fragments recovered in step 3 were sequenced. The sequencing results showed that the DNA fragments recovered in step 3 (i.e. OsAA2The nucleotide sequence of the gene is shown in SEQ ID NO: 1.

[0053] Example 2: Acquisition of transgenic rice and identification of its heat resistance 1. Construction of recombinant plasmid 1. Recombinant plasmid pCAMBIA1300- OsAA2 Construction (1) Using the DNA fragment obtained in step 3 of Example 1 as a template, primer F: 5'- TCTAG ATGGCGACGCTCACCGTCCC-3' (underlined is restriction endonuclease Xba I recognition site) and primer R: 5'- GGATCC TCACTGCTCCTGACCCAGAAGA-3' (underlined is restriction endonuclease Bam PCR amplification was performed using a primer pair consisting of the recognition site of HI, and then a 966 bp DNA fragment A was recovered.

[0054] (2) Use restriction endonucleases Bam The pCAMBIA1300 vector was digested with HI and XbaI to recover a vector backbone of about 15 kb.

[0055] (3) Connect DNA fragment A and vector backbone using infusion ligase to obtain recombinant plasmid pCAMBIA1300- OsAA2 .

[0056] The recombinant plasmid pCAMBIA1300- OsAA2 The sequencing results showed that the recombinant plasmid pCAMBIA1300- OsAA2 To insert the restriction endonuclease of pCAMBIA1300 vector Bam The small DNA fragment between HI and XbaI was replaced with the DNA molecule shown in SEQ ID NO: 1 to obtain a recombinant plasmid.

[0057] Recombinant plasmid pCAMBIA1300- OsAA2 The protein OsAA2 shown in SEQ ID NO: 2 is expressed.

[0058] 2. Recombinant plasmid pTCK303- OsAA2 Construction (1) Using the DNA fragment obtained in step 3 of Example 1 as a template, primers pTCK303- OsAA2 -F: 5'-GG GGTACCACTAGT ACACAAAGTTGGCACATT-3' (single underline is restriction endonuclease Kpn I recognition site, double underlined for restriction endonuclease Spe I recognition site) and primers pTCK303- OsAA2 -R:5'-CG GGATCCGAGCTC AGCATCCTCTCGTAGTC-3' (single underline is restriction endonuclease Bam The recognition site of HI, the double underlined one is the restriction endonuclease Sac PCR amplification was performed using a primer pair consisting of a recognition site of I, and then a DNA fragment 1 of about 458 bp was recovered.

[0059] (2) Connect DNA fragment 1 and T vector (Takara) to obtain recombinant plasmid T- OsAA2 -C.

[0060] (3) Use restriction endonucleases Spe I and Sac I digested the recombinant plasmid T- OsAA2 -C, recovery of DNA fragment 2.

[0061] (4) Use restriction endonucleases Spe I and Sac I was used to digest the pTCK303 vector and recover the vector backbone 1 of about 14.6 kb.

[0062] (5) Connect DNA fragment 2 and vector backbone 1 to obtain an intermediate vector.

[0063] (6) Use restriction endonucleases Kpn I and Bam HI digested recombinant plasmid T- OsAA2 -C, recovery of DNA fragment 3.

[0064] (7) Use restriction endonucleases Kpn I and Bam The intermediate vector was digested with HI to recover the vector backbone 2 of about 15.0 kb.

[0065] (8) Connect DNA fragment 3 and vector backbone 2 to obtain recombinant plasmid pTCK303- OsAA2 .

[0066] The recombinant plasmid pTCK303- OsAA2 The sequencing results showed that the recombinant plasmid pTCK303- OsAA2 To insert the restriction endonuclease of pTCK303 vector Spe I and Sac I recognition sequence between the small DNA fragments replaced by nucleotide sequence SEQ ID NO: 1 from 497 to 926 from the 5' end of the DNA molecule, restriction endonuclease Kpn I and BamThe small DNA fragment between the HI recognition sequences is replaced with a DNA molecule having a nucleotide sequence of SEQ ID NO: 3 (the DNA molecule shown in SEQ ID NO: 3 is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 from positions 497 to 926 from the 5' end), to obtain a recombinant plasmid.

[0067] 2. Obtaining recombinant Agrobacterium 1. Recombinant plasmid pCAMBIA1300- OsAA2 Introduce Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, named EHA105 / pCAMBIA1300- OsAA2 .

[0068] 2. Recombinant plasmid pTCK303- OsAA2 Introduce Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, named EHA105 / pTCK303- OsAA2 .

[0069] 3. Obtaining genetically modified rice 1. T3 generation homozygous transformation OsAA2 Obtaining genetically modified rice EHA105 / pCAMBIA1300- OsAA2 Transformed to Nipponbare, and obtained T0 generation pseudotransformation OsAA2 Genetic rice; then self-pollinate to obtain T3 generation homozygous transgenic rice OsAA2 Genetic rice. The method for screening positive seedlings is as follows: extract genomic DNA of the rice seedlings to be tested and use it as a template, use a primer pair consisting of 5'-TCTAGAATGGCGACGCTCACCGTCCC-3' and 5'-GGATCCTCACTGCTCCTGACCCAGAAGA-3' to perform PCR amplification to obtain PCR amplification products; then make the following judgment: if a PCR amplification product contains only a DNA fragment of about 966bp, the rice seedling corresponding to the PCR amplification product is a homozygous positive seedling.

[0070] 2. T3 generation silence OsAA2 Obtaining genetically modified rice EHA105 / pTCK303- OsAA2 Transformed to Nipponbare to obtain T0 generation pseudo-silent strain; then self-fertilized to obtain T3 generation silent strain OsAA2Genetic rice. The method for screening positive seedlings is as follows: extract genomic DNA of the rice seedlings to be tested and use it as a template, use a primer pair consisting of 5'-GGGGTACCACTAGTACACAAAGTTGGCACATT-3' and 5'-CGGGATCCGAGCTCAGCATCCTCTCGTAGTC-3' to perform PCR amplification to obtain PCR amplification products; then make the following judgment: if a certain PCR amplification product contains only a DNA fragment of about 458bp, the rice seedling corresponding to the PCR amplification product is a homozygous positive seedling.

[0071] 4. Real-time fluorescence quantitative PCR detection of transgenic rice OsAA2 Relative expression level of genes 1. Transform each T3 generation homozygous cells grown for 7 days into OsAA2 The genetically modified rice seedlings were stored in liquid nitrogen to obtain the corresponding test samples. OsAA2 The genetically modified rice seedlings were stored in liquid nitrogen to obtain the corresponding samples to be tested. The 7-day-old Nipponbare seedlings were stored in liquid nitrogen to obtain the corresponding samples to be tested.

[0072] 2. The total RNA of the sample to be tested was extracted by Trizo1 method, and then the first-strand cDNA was reverse transcribed using a reverse transcription kit (Takara); the cDNA was diluted 10 times with sterile water as a template for real-time quantitative PCR detection. OsAA2 The relative expression level of genes ( OsACTIN1 gene as the internal reference gene).

[0073] Detection OsAA2 The primers for the gene were 5′-AGTGCTTACCGCTATGAGGGGC-3′ and 5′-GCAGCGTGGAAAGGAACTCG-3′.

[0074] Detection OsACTIN1 The primers for the gene were 5′-CATCTCTCAGCACATTCCAGCAG-3′ and 5′-AGGAGGACGGCGATAACAGC-3′.

[0075] Some test results can be found in Picture 2 (WT is Nipponbare, OE7, OE8 and OE11 are all T3 generation homozygous transgenic OsAA2 Genetic rice, RNAi7, RNAi8 and RNAi21 are all silenced in the T3 generation OsAA2 The results showed that compared with WT, each T3 generation homozygous transgenic rice OsAA2 Genetic rice OsAA2 The relative expression levels of the genes were significantly increased. OsAA2 The gene rice lines were named OE7, OE8 and OE11. Compared with Nipponbare, each T3 generation was silent OsAA2 Genetic rice OsAA2 The relative expression levels of the genes were significantly reduced, and three of the T3 generations were silenced. OsAA2 The gene rice lines were named RNAi7, RNAi8 and RNAi21.

[0076] 5. Identification of heat tolerance of transgenic rice 1. Heat resistance identification during germination The rice seeds to be tested are T3 generation seeds of OE8, T3 generation seeds of OE11, T3 generation seeds of RNAi7, T3 generation seeds of RNAi8 or Nipponbare seeds.

[0077] The experiment was repeated three times to obtain the average value. The steps for each repetition were as follows: ① Take the rice seeds to be tested, wash them with 50% (v / v) sodium hypochlorite solution for 20 minutes, and then wash them with sterile water 6 times.

[0078] ②After completing step ①, 20 rice seeds to be tested were randomly divided into two groups: a heat treatment group and a normal group, with 10 seeds in each group. Each group was treated as follows: Heat treatment group: 50℃ treatment for 3h, followed by alternating light and dark culture at 28℃ for 7 days to obtain the rice seedlings to be tested; Normal group: treated at 28°C for 3 h, then cultured at 28°C with alternating light and dark for 7 days to obtain the rice seedlings to be tested.

[0079] ③After completing step ②, observe the growth status of the rice seedlings to be tested.

[0080] The growth status of some rice seedlings to be tested is shown in Picture 3 (WT is Nipponbare, 50℃ 3h is the heat treatment group, and 28℃ is the normal group).

[0081] ④After completing step ③, count the length of the above-ground part and the root length of the rice seedlings to be tested.

[0082] The statistical results are shown in Picture 4 (WT is Nipponbare, Heat treatment is heat treatment group, Normal is normal group).

[0083] The results showed that if the germination period was not subjected to high temperature treatment (i.e., the whole process was cultured at 28°C), the T3 generation homozygous transgenic OsAA2 Genetic rice (OE8 and OE11), T3 generation silencing OsAA2There was no significant difference in growth status, length of aboveground part and root length between the transgenic rice (RNAi7 and RNAi8) and Nipponbare; if the germination period was treated with high temperature (i.e., 50℃ for 3h and then cultured at 28℃): compared with Nipponbare, the T3 generation homozygous transgenic rice OsAA2 The growth status of the genetically modified rice (OE8 and OE11) is good, and the T3 generation is silent OsAA2 The growth state of gene rice (RNAi7 and RNAi8) is poor (manifested as obvious no growth); compared with Nipponbare, the T3 generation is silent OsAA2 The length of the aboveground part and the root length of the transgenic rice (RNAi7 and RNAi8) were significantly reduced; Nipponbare and T3 homozygous transgenic rice OsAA2 There was no significant difference in the length of the aboveground part and the length of the root between the genetically modified rice (OE8 and OE11).

[0084] 2. Heat resistance identification at the seedling stage The rice seeds to be tested are T3 generation seeds of OE8, T3 generation seeds of OE11, T3 generation seeds of RNAi7, T3 generation seeds of RNAi8 or Nipponbare seeds.

[0085] The experiment was repeated three times to obtain the average value. The steps for each repetition were as follows: ① Take the rice seeds to be tested, wash them with 50% (v / v) sodium hypochlorite solution for 20 minutes, wash them with sterile water 6 times, and then culture them at 28°C with alternating light and dark for 7 days to obtain the rice seedlings to be tested, that is, the rice seedlings to be tested before heat treatment.

[0086] ② After completing step ①, 40 rice seedlings to be tested with the same growth status were transplanted to a 96-well plate, cultured at 28° C. for 1 day with alternating light and dark, and then treated at 50° C. for 11 h to obtain the heat-treated rice seedlings to be tested.

[0087] ③After completing step ②, take the rice seedlings to be tested before heat treatment or the rice seedlings to be tested after heat treatment, and measure the malondialdehyde content and relative conductivity.

[0088] Test results see Picture 5 (WT is Nipponbare, Heat treatment is the rice seedlings to be tested after heat treatment, Normal is the rice seedlings to be tested before heat treatment, and MDA content is the malondialdehyde content).

[0089] The results showed that if the seedlings were not subjected to high temperature treatment (i.e., not treated at 50°C for 11 h), the homozygous transgenic plants in the T3 generation would OsAA2 Genetic rice (OE8 and OE11), T3 generation silencing OsAA2 There was no significant difference in the malondialdehyde content and relative conductivity between the transgenic rice (RNAi7 and RNAi8) and Nipponbare. If the seedlings were treated with high temperature (i.e., 50°C for 11 h), the T3 generation homozygous transgenic rice was significantly higher than that of Nipponbare. OsAA2 The malondialdehyde content of genetically modified rice (OE8 and OE11) was significantly reduced, and the T3 generation was silent OsAA2 The malondialdehyde content and relative conductivity of genetic rice (RNAi7 and RNAi8) were significantly increased.

[0090] The above results indicate that increasing the expression level of protein OsAA2 in Nipponbare rice can improve the heat tolerance of rice; the improvement in heat tolerance is manifested as: a significant increase in the length of the aboveground part, a significant increase in the root length, a significant decrease in the malondialdehyde content and / or a significant decrease in relative conductivity.

[0091] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Application of protein OsAA2, either S1) or S2): S1) Regulates heat tolerance in rice; S2) Cultivation of transgenic rice with altered heat tolerance; The amino acid sequence of the protein OsAA2 is shown in SEQ ID NO:

2.

2. Use of a nucleic acid molecule encoding the protein OsAA2 according to claim 1, as S1) or S2): S1) Regulates heat tolerance in rice; S2) Breeding transgenic rice with altered heat tolerance.

3. The use according to claim 1 or 2, characterized in that: The regulating the heat tolerance of rice is to increase the heat tolerance of rice or to reduce the heat tolerance of rice; The cultivation of transgenic rice with altered heat resistance is cultivation of transgenic rice with improved heat resistance or cultivation of transgenic rice with reduced heat resistance.

4. A method for cultivating transgenic rice beta, comprising the following steps: increasing the expression level and / or activity of the protein OsAA2 in the starting rice to obtain transgenic rice beta; compared with the starting rice, the heat resistance of the transgenic rice beta is improved; the amino acid sequence of the protein OsAA2 is shown in SEQ ID NO:

2.

5. The method according to claim 5, characterized in that: The increasing of the expression level and / or activity of the protein OsAA2 in the starting rice is achieved by introducing a nucleic acid molecule encoding the protein OsAA2 into the starting rice.

6. A method for cultivating transgenic rice B, comprising the following steps: inhibiting the expression level and / or activity of protein OsAA2 in a starting rice to obtain transgenic rice B; compared with the starting rice, the heat resistance of transgenic rice B is reduced; the amino acid sequence of the protein OsAA2 is shown in SEQ ID NO:

2.

7. The method according to claim 6, characterized in that: The inhibition of the expression level and / or activity of the protein OsAA2 in the starting rice is achieved by introducing a substance that inhibits the expression of a nucleic acid molecule encoding the protein OsAA2 into the starting rice; Furthermore, the substance that inhibits the expression of the nucleic acid molecule encoding the protein OsAA2 is a specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule; The specific DNA molecule includes a sense segment, an antisense segment and a spacer segment between them; The sense fragment is the DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end; The antisense fragment is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end.

8. Rice breeding method A or rice breeding method B; The rice breeding method A comprises the following steps: increasing the content and / or activity of the protein OsAA2 in rice, thereby improving heat resistance; The rice breeding method B comprises the following steps: reducing the content and / or activity of the protein OsAA2 in rice, thereby reducing heat resistance; The amino acid sequence of the protein OsAA2 is shown in SEQ ID NO:

2.

9. A specific DNA molecule, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule; The specific DNA molecule includes a sense segment, an antisense segment and a spacer segment between them; The sense fragment is the DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end; The antisense fragment is the reverse complementary sequence of the DNA molecule shown in SEQ ID NO: 1 from position 497 to position 926 from the 5' end.

10. Use of the specific DNA molecule according to claim 9, an expression cassette containing the specific DNA molecule, or a recombinant plasmid containing the specific DNA molecule in reducing the heat tolerance of rice or cultivating transgenic rice with reduced heat tolerance.

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